Figure legends
FIGURE 1.  Neonatal treatment with MK-801 selectively reduces synaptic strength in the MPP – DG synapse. (a) Schematic representation of the EC – DG circuit and placement of stimulation and recording electrodes for extracellular recordings. Stimulation of the lateral perforant path (LPP) and the resulting LPP fEPSP were acquired in the outer one-third section of the molecular layer of the DG (green axons). Medial perforant path or MPP fEPSPs were acquired in the middle one-third of the molecular layer of the DG (red axons). (b)Pharmacological sensitivity of the LPP fEPSPs and the MPP fEPSP to different metabotropic glutamate receptors. Upper panel (green traces): LPP fEPSP are sensitive to activation of group III mGluRs with L-AP4 (20 µM) and insensitive to stimulation of group II mGluRs with DCG-IV (5 µM). Lower panel (red traces): MPP fEPSP are insensitive to activation of group III mGluRs. The opposite occurs in the presence of group II mGluRs agonists. The pharmacological sensitivity of the fEPSPs to the different mGluRs was rigorously used as an inclusion criterion for all the experiments included in this study. (c) Input-output graphs of the LPP fEPSPs and (d) the MPP fEPSPs in response to increasing current pulses (100 µA steps at 0.067 Hz). Neonatal treatment with MK-801 did not alter the magnitude of the LPP fEPSP (upper trace and green symbols in panel c) but reduced the magnitude of the MPP fEPSP (upper trace and red symbols in panel d), **P  < 0.01.(e) Left panel: input-output curve of the LPP fiber volleys (FV) in response to increasing current pulses; right panel: coupling analysis of the FV vs. LPP fEPSP slope, showing no difference in synaptic strength. (f) Left panel: input-output curve of the MPP FV in response to increasing current pulses, showing decreased presynaptic excitability in the MK-801 group; right panel: coupling analysis of FV vs. MPP fEPSP slope. * P  < 0.05 and **P  < 0.01. For LPP:  n  = 9 slices / 6 animals for the control group and n  = 10 slices / 6 animals for the MK-801 group. For MPP: 8 slices / 6 animals for the control group and n  = 10 slices / 6 animals for the MK-801 group.
FIGURE 2. Neonatal treatment with MK-801 reduces presynaptic neurotransmitter release at the LPP and the MPP. (a) Violin plots contrasting the paired-pulse facilitation values (PPF; ISI 60 ms) of the LPP (n = 28 slices / 15 animals; green symbols) and the MPP (n = 29 slices / 15 animals; red symbols) synapses in the control condition. LPP PPF was greater than MPP PPF, ***P < 0.001.(b) Representative traces showing the pharmacological sensitivity of the LPP and the MPP synapses. LPP PPF decreased in the presence of L-AP4 (20 µM, upper traces), whereas the MPP PPF was abolished by perfusion of DCG-IV (5 µM; lower traces). (c) Time course of the LPP PPF exploring different ISI (40, 60, 100, 200, and 500 ms). Neonatal treatment with MK-801 increased the LPP PPF. *P< 0.05. For LPP PPF: n = 8 slices / 6 animals for control and n = 9 slices / 7 animals for MK-801. (d)Representative PPF of the LPP synapses contrasting the values of control and MK-801-treated slices. (e) Time course and (f)representative traces for MPP PPF in control (red symbols and traces) and MK-801-treated (black symbols and traces) slices. MPP PPF: n= 9 slices / 7 animals for each condition. *P < 0.05.
FIGURE 3. Neonatal treatment with MK-801 impairs the induction of CB1R-dependent LTD at the MPP – DG synapse. (a)Representative traces of control (upper traces) and the CB1R antagonist, AM251 treatment (5 µM, lower traces) in baseline, post-LFS, and the presence of DCG-IV (5 µM). (b) Time course graph of fEPSP slope in response to LFS (900 pulses at 3 Hz) in the MPP. In the control condition (black symbols), LFS induces LTD. Perfusion of AM 251 (5 µM, white symbols) LFS prevented the induction of LTD. (c) Representative traces of the control (upper black traces) and MK-801-treated group (lower red traces) in baseline, post-LFS, and the presence of DCG-IV (5 µM). (d) Time course graph of LTD. In the MK-801-treated group, LFS failed to induce LTD; an abnormal potentiation (red symbols) was observed that was sensitive to DCG-IV (5 µM). (e) Bar graph summarizing the magnitude of normalized fEPSP slopes at 90 min after LFS and in the presence of DCG-IV in the control (symbols and black bars), AM-251 (symbols and white bars), and MK-801 (symbols and red bars) groups. *P< 0.05. Each symbol within the bars represents one independent experiment; bars represent the media ± SEM. (f) Bar graph showing the changes in PPR before and after LFS. The control group exhibited increased PPR after LFS, suggesting a reduction in presynaptic release at the MPP. *P < 0.05; n = 6 slices / 6 animals for control and MK-801; n = 4 slices / 4 animals for AM 251.(g) Cumulative probability distribution chart of normalized fEPSPs post-LFS in the control (black line), AM-251 (gray line), and MK-801 (red line) groups. The individual values for this analysis (bins configured at 0.8 value) correspond to 10 to 90 minutes of the time course in panels b and c .
FIGURE 4. Neonatal treatment with MK-801 reduces the functional expression of CB1R but not the synthesis of 2-AG at the MPP – DG synapse. (a) Representative traces of control and MK-801 fEPSPs during baseline (1), 90 min post-WIN 55,212-2 (2), and DCG-IV (3). (b) Time course graph of normalized fEPSP slope in response to the perfusion of the CB1R agonist, WIN 55,212-2 (5 µM for 15 min), in control and MK-801-treated groups (black and red symbols, respectively). (c) Representative traces and(d) time course graph of normalized fEPSPs in the presence of the cholinesterase inhibitor physostigmine (10 µM for 15 min) in control and MK-801-treated groups (black and red symbols, respectively). Slices of the MK-801-treated group exhibited synaptic depression in response to physostigmine perfusion. (e) Bar graph contrasting the synaptic depression elicited with WIN 55,212-2 in control vs. MK-801-treated groups. *P < 0.05, n = 6 slices / 5 animals for control and n = 6 slices / 6 animals for MK-801. (f) Bar graph contrasting the magnitude of synaptic depression in response to physostigmine. The MK-801-treated group (red bar) exhibited reduced synaptic depression; however, this difference lacked statistical significance compared to the control group (black bar). n = 6 slices / 6 animals for control and n = 5 slices/ 5 animals for MK-801. (g) Bar graph contrasting the magnitude of synaptic depression in the MK-801-treated groups in response to WIN 55,212-2 or physostigmine. The synaptic depression induced by physostigmine is similar to the magnitude of depression induced by activation of CB1R with WIN, suggesting that 2-AG synthesis is not affected by neonatal treatment with MK-801.
FIGURE 5. Pharmacological inhibition of monoacylglycerol lipase (MAGL) enzyme restores the impaired CB1R-dependent LTD at the MPP – DG synapse. (a) Representative fEPSP traces from MK-801-treated slices (red traces) or those preincubated with JZL 184 (blue traces) in the conditions indicated by the numbers. (b)Time course graph of normalized fEPSPs from MK-801-treated slices in the presence of the irreversible inhibitor of monoacylglycerol lipase (MAGL JZL 184, 1 µM; blue symbols). LFS induced stable LTD in the presence of JZL 184 MK-801-treated slices. (c) Bar graph. Perfusion of JZL 184 rescues LTD at the MPP – DG synapse. *P < 0.05; n = 6 slices / 6 animals for MK-801-treated group and n = 5 slices / 5 animals for MK-801 + JZL 184 group. (d) Bar graphs showing restored LTD is accompanied by increased PPR. * P < 0.05. (e)Cumulative probability distribution plot of the fEPSP slope values after LFS (red line) or LFS + JZL (blue line) in MK-801-treated slices. The individual values for this analysis (bins configured at 0.8 value) correspond to 10 to 90 minutes of the time course in panel b.
FIGURE 6. Neonatal treatment with MK-801 impairs the TBS-induced LTP at the LPP – DG synapse. (a) Representative traces of fEPSP from control (black traces) and MK-801 (green traces) in the conditions indicated by the numbers. (b) Time course of normalized fEPSP slope from control and MK-801 in response to TBS. Arrowhead indicates the delivery of the TBS train (10 bursts at 5 Hz; each burst consisted of 5 pulses at 100 Hz). Inset bar graph comparing the post-tetanic potentiation decay calculated by adjusting a best-fit single exponential decay function. The PTP dropped faster in the MK-801-treated group. *P < 0.05. (c) Bar graph contrasting the magnitude of PTP, LTP, and the effect of L-AP4 (20 µM) on the LPP synapse. *P < 0.05; n = 7 slices / 6 animals for control and n = 7 slices / 7 animals for MK-801. (d) Heatmaps showing the magnitude of LTP in each slice from both experimental groups.(e) Cumulative probability distribution plot of the fEPSP slope values. The individual values for this analysis (bins configured at 0.8 value) correspond to 10 to 90 minutes of the time course in panel b.
FIGURE 7. Neonatal treatment with MK-801 does not interfere with the induction of TBS-induced LTP at the MPP – DG synapse. (a)Representative traces of fEPSP from control (black traces) and MK-801 (red traces) in the conditions indicated by the numbers. (b)Time course of normalized fEPSP slope in response to TBS. Inset bar graph comparing the post-tetanic potentiation decay calculated by adjusting a best-fit single exponential decay function. No difference was found in either group. (c) Bar graph contrasting the magnitude of PTP, LTP, and the effect of DCG-IV (5 µM). n = 8 slices / 7 animals for control and n = 7 slices / 7 animals for MK-801.(d) Heatmap showing the magnitude of LTP in each slice from both experimental groups. (e) Cumulative probability distribution plot of the fEPSP slope values.
FIGURE 8. Neonatal treatment with MK-801 impairs frequency-dependent synaptic filtering at the LPP. (a1-a3, left panels)Scatter plot showing the evoked fEPSPs slope from control (black symbols) and MK-801-treated (green symbols) groups in response to 5, 20, and 50 Hz trains. In control slices, 5 Hz induced synaptic facilitation throughout the stimulation train, 20 Hz caused a mild synaptic depression at the end of the train, and 50 Hz induced massive attenuation of the synaptic response (horizontal dashed lines represent facilitation value vs. S1). In the MK-801-treated slices, frequency-dependent filtering was impaired at 5 Hz and 50 Hz since synaptic facilitation was increased. *P < 0.05. Train at 5 Hz: n = 9 slices/animals for control and n = 7 slices / 7 animals for MK-801. Train at 20 Hz and 50 Hz: n = 8 slices / 6 animals for control and n = 8 slices / 7 animals for MK-801. (b1-b3) Representative traces in response to trains at 5 Hz, 20 Hz, and 50 Hz. (c1-c3)Over-imposed traces showing the first synaptic response (S1) vs. the tenth response (S10) in the three frequencies examined. (d-f)Bar graphs contrasting the magnitude of the fEPSP amplitude (measured in mV) from S1 and S10 in both experimental conditions at the three frequencies tested.
FIGURE 9. Neonatal treatment with MK-801 impairs spatial discrimination in male rats. (a) Schematic representation of the object spatial pattern separation (OPS) task. The task comprised a learning trial (T1) and a discrimination trial (T2). It was performed in a circular arena with two equidistant identical objects (40 cm) placed in five positions (P), with 6 cm between positions. These positions were designated both left and right from the center of the arena. In T1, the animals were exposed to two objects placed in P1. In T2, both objects were placed in P1 (null displacement), or one was placed in P2, P3, P4, or P5 (maximal displacement). The discrimination index (DI) was indicative of performance in this task. All animals were evaluated in all P values with intervals of two days. Objects in green or gray prism form represent identical par objects. (b) Bar graph summarizing the DI values obtained from control (black bars) and MK-801-treated (blue bars) animals during the OPS task. MK-801-treated animals could not distinguish the displacement of one object to P3, indicating impaired spatial discrimination. *P < 0.05, n = 10 for control and n = 12 for MK-801. (c) Heatmap showing all DI values for each animal from both experimental conditions. While the color scale in the P5 columns is similar in control and MK-801-treated animals, the color pattern in the P3 columns differs between experimental conditions, indicating reduced DI values in MK-801-treated animals. (d)Line chart summarizing DI performance from control (black line) and MK-801 (blue line) in function of the magnitude of displacement of one object in T2. In control animals, when the magnitude of displacement of one object is decreased, the cognitive demand of pattern separation is theoretically increased, which supports the spatial discrimination of the position of objects. However, when the magnitude of displacement of one object is maximal (24 cm), MK-801-treated animals exhibit similar behavioral performance to control animals. When the cognitive demand of pattern separation increases, the MK-801-treated animals exhibit impaired performance in spatial discrimination.